The establishment of precise neuronal connectivity during development requires that neurons respond to a myriad of guidance cues, both attractive and repulsive, as they extend toward their targets and ultimately establish final connectivity patterns. Importantly, many of these guidance cues and their receptors are also expressed in the adult nervous system where they may play important roles in the assembly and regulation of neuronal circuits. In addition, the expression patterns of several of these cues and their receptors show changes in response to neuronal injury. In this proposal we will explore how semaphorins, a large protein family which includes many members capable of functioning as repulsive neuronal guidance cues, direct the formation of mammalian central and peripheral neural connections during development and in the adult. Secreted semaphorins are robust neuronal repellents in vitro and in vivo, and we have shown during the initial funding period of this grant that certain secreted semaphorins and their receptors the neuropilins are indeed required in vivo for the establishment of normal connectivity in a wide variety of neural systems. Recent genetic and biochemical analyses have also shown that members of the plexin family of transmembrane proteins serve as signaling subunits of secreted semaphorin receptors. Thus neuropilins are the ligand binding subunits and plexins are the signaling subunits of holoreceptors for the secreted semaphorins. Using several mouse genetic models generated as part of our studies, and employing certain new mouse mutants, we propose here to provide a comprehensive understanding of how neuropilins, plexins and their six secreted semaphorin ligands Sema3A, B, C, D, E, and F (all defined as Class 3 semaphorins) participate in the establishment of neuronal connectivity in both the CNS and PNS. In all, these studies will help us move beyond our current, incomplete, understanding of functional ligand-receptor relationships between secreted semaphorins and their receptors. They will provide insight into how secreted semaphorins, acting as repellents and possibly as attractants, work in concert to regulate neuronal migration, morphology, pathway formation, and target selection in the embryonic and postnatal nervous system. Finally, these studies will begin to address how class 3 semaphorins and their receptors function once neural circuits are established to modulate neuron morphology and synapse function.